Controllable Transcription

Abstract

The present invention relates to a stable method for introducing at least one inducible cassette into a cell, and permitting controllable transcription from within that inducible cassette. The method may be used for any cell type, from any eukaryotic organism, but has a particular application in the introduction of inducible cassettes into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted in such a way to ensure that the genetic material it contains is not silenced or subject to negative influences from the insertion site, and transcription of the genetic material is controlled.

1 . A method for production of adipocytes from pluripotent stem cells, comprising the steps of:
 a) inserting a gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and   b) inserting a gene encoding an adipocyte lineage master regulator operably linked to an inducible promoter into a second genetic safe harbour site;   wherein said inducible promoter is regulated by the transcriptional regulator protein;   wherein said first and second genetic safe harbour sites are different; and   wherein the pluripotent stem cell is an animal cell.   
     
     
         2 . The method of  claim 1 , wherein the animal cell is a mammalian, marsupial, non-human primate, camelid, or livestock animal cell. 
     
     
         3 . The method of  claim 1 , wherein the animal cell is from a livestock animal. 
     
     
         4 . The method of  claim 1 , wherein the animal cell is from a pig or from cattle. 
     
     
         5 . The method of  claim 1 , wherein the method is for programming of the pluripotent stem cell into a mature cell. 
     
     
         6 . The method of  claim 1 , wherein the method is for programming of the pluripotent stem cell into an adipocyte. 
     
     
         7 . The method of  claim 1 , wherein the transcriptional regulator protein is selected from the group consisting of: a reverse tetracycline transactivator protein (rtTa), a Tetracycline repressor (TetR), a VgEcR synthetic receptor, a hybrid transcriptional regulator protein comprising a DNA binding domain from yeast GAL4 protein, a truncated ligand binding domain from the human progesterone receptor, and an activation domain from human NF-κB. 
     
     
         8 . The method of  claim 7 , wherein activity of the rtTA is controlled by tetracycline or a derivative thereof. 
     
     
         9 . The method of  claim 7 , wherein activity of the rtTA is controlled by doxycycline. 
     
     
         10 . The method of  claim 1 , the inducible promoter includes a Tet Responsive Element (TRE). 
     
     
         11 . The method of  claim 1 , wherein the first and second genomic safe harbour sites are selected from any two of a hROSA26 locus, a AAVS1 locus, a CLYBL gene, or a CCR5 gene. 
     
     
         12 . The method of  claim 1 , wherein additional genetic material is inserted at the first and/or second genomic safe harbour sites. 
     
     
         13 . The method of  claim 12 , wherein the additional genetic material is selected from the group consisting of:
 a) suicide gene;   b) selectable marker;   c) reporter gene; and   d) gene for a non-coding RNA.   
     
     
         14 . A cell obtained by the method of  claim 1 . 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled)